This invention relates to plant transformation constructs containing genes that encode enzymes that can open aromatic rings. It also relates to the insertion of these genes into plant cells, and to the regeneration of those cells into transgenic plants that can degrade aromatic compounds in soil by opening an aromatic ring.
As a result of spills, accidents, construction, the use of outdoor storage ponds, poorly controlled manufacturing processes, and the deliberate use of herbicides and pesticides, aromatic compounds have found their way into soil in concentrations considered to be environmentally hazardous. Not only are these compounds often malodorous and the source of health problems, but, in high concentrations, they can represent a fire hazard. Moreover, the compounds are not readily biodegraded and persist for many years.
Over time, these aromatic compounds can seep into the ground water from which many municipalities in the United States draw their drinking water. Because the compounds tend to accumulate in fatty tissue and may be hazardous, it is necessary to remove them from the water and prevent them from entering the food chain. As a consequence, there is considerable pressure from government agencies to remove these aromatic compounds from the soil, where they are more concentrated, before they can enter the ground water. This can be an expensive process because the compounds are usually present in very low concentrations.
The removal and destruction of these compounds in soil is a formidable task. If the compounds are volatile they can sometimes be stripped from the soil with air or, if they are soluble, they can sometimes be removed by solvent extraction; but, after removal, it is still necessary to degrade or destroy them. Degradation in the soil is difficult as it requires a chemical reaction. Since the aromatic compounds are usually present in small concentrations, and various other reactive organic compounds are also present in the soil, a large quantity of chemicals is required for effective treatment. Incineration of the soil is effective in destroying the aromatic compounds but is very expensive and is impractical when the aromatic compounds are present in low levels.
Certain bacteria and fungi are known to produce enzymes that are capable of degrading aromatic compounds, including chlorinated aromatic compounds. While the use of bacteria has some advantages over other methods of degrading aromatic compounds in soil, bacterial remediation is not without its problems. (See, for example, "Bioengineering Issues Related to in situ Remediation of Contaminated Soils and Groundwater," by Perry L. McCarty, Environmental Biotechnology, ed. by Gilbert S. Omenn, Plenum Press (1988)). First, it is usually necessary to use aerobic bacteria because anaerobic bacteria work too slowly and are not effective near the surface of the soil where oxygen penetrates. On the other hand, aerobic bacteria are effective only near the surface of the soil, and, in order to increase their penetration into the soil, it is necessary to aerate the soil, which adds to the expense of the process. These bacteria are specially selected and propagated and are usually not only quite expensive, but are also required to be used in large amounts in order to effectively treat a contaminated area. Moreover, once released into the environment, it is difficult to contain them and prevent them from spreading out to other areas. As a result, the use of bacteria creates regulatory problems and can arouse public opposition which further adds to the cost of treating contaminated soil.
What is needed is an inexpensive, safe, and effective method of degrading aromatic compounds in soil without the addition of chemicals or microorganisms to the soil. Until now, no such method has been found.